The rapidly increasing resistance rate to antiviral agents necessitates new combination drugs with different mechanism of action as well as novel drugs acting on newly discovered targets. As viral diseases are frequently associated with several kinds of viruses, the research on novel antiviral agents with multiple mechanisms of action on as many viruses as possible is of particular importance for the improvement of treatment outcomes.
RNA molecules can adopt a wide variety of conformations and perform a range of cellular functions. This is achieved due to their structure allowing them to form specific RNA-RNA, RNA-DNA or RNA-protein interactions. Achievements in the study of these RNA interactions permitted to develop novel methods of treatment of various disorders. So far the most attention has been received by therapeutic RNAs which can be classified as gene inhibitors, gene amenders, protein inhibitors and immunostimulatory RNAs. (Bruce A. Sullenger & Eli Gilboa, Emerging clinical applications of RNA, Nature, 2002, 418, July 11, pp. 252-258)
Gene inhibitors are represented by complementary RNAs which specifically recognize their target transcripts by forming base pairs with them in a sequence-dependent manner. They are also termed antisense RNAs. The formation of this RNA duplex is believed to lead to the degradation of the target RNA or the inhibition of its translation. Further discovery that certain RNAs can perform catalysis of RNA hydrolysis led the development of a novel class of therapeutic RNAs called trans-cleaving ribozymes. Such ribozymes bind substrate RNAs through base-pairing interactions, cleave the bound target RNA, thus releasing the cleavage products. (Guerrier-Takada, C., Gardiner, K., Marsh, T., Pace, N. & Altman, S, The RNA moiety of ribonuclease P is the catalytic subunit of the enzyme, Cell, 1983, 35, pp. 849-857) Several phase I and phase II trials have been initiated using trans-cleaving ribozymes in a small number of patients with infectious diseases. (Wong-Staal, F., Poeschla, E. M. & Looney, D. J., A controlled, Phase I clinical trial to evaluate the safety and effects in HIV-1 infected humans of autologous lymphocytes transduced with a ribozyme that cleaves HIV-1 RNA, Hum. Gene Ther. 1998, 9, pp. 2407-2425) Unfortunately, as shown in these trials, a sustained long-term effect has not been achieved. (Bruce A. Sullenger & Eli Gilboa, Emerging clinical applications of RNA, Nature, 2002, 418, July 11, pp. 252-258) The critical factors determining the success of these synthetic ribozyme efficacy trials include the ability to deliver ribozymes efficiently into the appropriate cells in vivo, and the level and duration of target-gene inhibition that is required to slow the rate of disease progression. To benefit patients with chronic diseases such as cancer, HIV-infection, and hepatitis C, long-term and high-level inhibition of target transcripts is required. In practice this may be difficult to achieve, especially when targeting highly expressed viral RNAs. Protein inhibitors are represented by artificially-produced RNAs which can adopt complex secondary structures that allow them to bind target proteins with high affinity and specificity. (Daley D. T. A., Luscombe N. M., Berman H. M. and Thornton J. M., Protein-RNA interactions: a structural analysis. Nucleic Acids Research, 29, 4, pp. 943-954) For selection of these biologically active RNAs representing oligonucleotides 15-35 nucleotides long, a number of methods has been developed. Such oligonucleotides can specifically bind target proteins. (A. D. Keefe, S. Pai, A. Ellington, Aptamers as therapeutics, Nature Review, 2010, 9, pp. 537-550)
First of all, technologies of oligonucleotide synthesis have been developed, permitting synthetic ologinucleotides to bind a wide range of target proteins with high affinity. Target proteins include cytokines, proteases, kinases, cell surface receptors and cell adhesion molecules. Currently synthetic oligonucleotides are being developed which can be administered systemically into organism and bind blood targets (such as thrombin, IXa factor, von Willebrand factor) or cell surface targets such as epidermal growth factor receptors (EGFR). Oligonucleotides produced by chemical synthesis have a promising therapeutic potential and are currently evaluated in clinical trials for the treatment of diseases of eyes, blood, and cancer. (Keefe A. D., Schaub R. G., Aptamers as candidate therapeutics for cardiovascular indications, Curr. Opin. Pharmacol. 2008, Apr., 8(2), pp. 147-52, Epub Jan. 28, 2008; Barbas A. S., Mi J., Clary B. M., White R. R., Aptamer applications for targeted cancer therapy, Future Oncol., 2010, July, 6(7), pp. 1117-26)
Most of chemically synthesized oligonucleotides are subject to nuclease-mediated degradation by serum nucleases, renal filtration, uptake by the liver, spleen, and other tissues. Therefore, the half-life period of oligonucleotides which are not protected by modified nucleotides is not exceeding 2 minutes. Although it is quite a work-consuming process, nucleotides can be specifically modified and protected at the 3′ terminus against the action of serum nucleases. (Floege J. et al., Novel approach to specific growth factor inhibition in vivo antagonism of platelet-derived growth factor in glomerulonephritis by aptamers, American Journal of Pathology, 1999, 154, pp. 169-179; Beigelman, L. et al., Synthesis and biological activities of a phosphorodithioate analog of 2′,5′-oligoadenylate, Nucleic Acids Res., 1995, 23, pp. 3989-3994) This strategy permits to prolong the serum half-life of these oligonucleotides about 10-fold. To retard renal elimination oligonucleotides are conjugated with polyethylenglycol (PEG) with high molecular weight. In a mouse model non-conjugated oligonucleotides are eliminated from blood with a half-life of 5-10 min., while a 40 kDa PEG conjugates persist in circulation with a half-life of I day. (Burmeister, P. E. et al., Direct in vitro selection of a2′-O-methyl aptamer to VEGF. Chem. Biol., 2005, 12, pp. 25-33)
To date there are several chemically synthesized oligonucleotides that have progressed through clinical evaluation and are currently under review. In December 2004 the first chemically synthesized oligonucleotide Pegaptanib was approved by the US Food and Drug Administration for therapeutic use and is currently marketed by Pfizer and Eyetech as Macugen®. It is an oligonucleotide having a vascular endothelial growth factor (VEGF)-binding sequence of 27-nucleotides. It is administered at 6-week intervals by intravitreal injections for improvement in visual acuity in patients with age-related macular degeneration. (Chakravarthy, U. et al., Year 2 efficacy results of 2 randomized controlled clinical trials of pegaptanib for neovascular age-related macular degeneration, Ophthalmology, 2006, 113, e1-e25).
REG1 is an oligonucleotide having a 34-nucleotide IXa coagulation factor-binding sequence. Currently it is evaluated in phase 2 clinical trials. It shows rapid onset of anticoagulation in viva after intravenous administration, and rapid reversal of anticoagulation effect with return to initial level with antidote RB007. (Chan M. Y. et al., Phase 1b randomized study of antidote-controlled modulation of Factor IXa activity in patients with stable coronary artery disease circulation, 2008, 117, pp. 2865-2874)
ARC1905 is a 39-sequence oligonucleotide, currently evaluated in phase 1 clinical trials. It is used in combination with ranibizumab (which is a VEGF-specific monoclonal antibody fragment) for the treatment of age-related macular degeneration. (Biesecker G., et al., Derivation of RNA aptamer inhibitors of human complement C5, Immunopharmacology, 1999, 42, 1-3, pp. 219-230)
As most of these agents belong to protein inhibitors, their final success depends on their ability to compete with other classes of therapeutic agents, in particular, with monoclonal antibodies which are currently being evaluated along with the most of oligonucleotides in preclinical studies. Moreover, oligonucleotides are characterized by high specificity to target proteins. Each oligonucleotide is capable of inhibiting only one specific protein; this makes them very unreliable agents for the treatment of viral diseases. Viruses are known to develop resistance to specific inhibitors, therefore the perspective of use of such highly specific oligonucleotides for the treatment of viral diseases is doubtful.
Yeast-derived RNA molecules have been long used as therapeutic agents. (Zemskov V. M., Lidak M., Zemskov A. M., Mikstays U. Ya., Small RNA. Preparation, hydrolysis and its application in medicine, Riga, Zanatne, 1985, p. 191) However, in this case a′ sodium salt of yeast-derived RNA is used. It is not purified, and is characterized by high molecular weight heterogeneity (as it contains the whole spectrum of nucleotide components from dinucleotides to small transport-RNAs). It also possesses immuno-modulating properties. (Zemskov A. M., Perederiy V. G., Zemskov V. M., Bychkova N. G., Immunocorrective nucleic acid drugs and their clinical application, Kiev, Healthy, 1994, p. 232). Yeast-derived RNA is known to be used for wound healing. (Kulkarni et al., Ribonucleotide preparations and uses thereof—U.S. Pat. No. 5,712,256, Jan. 27, 1988) However, in this case, products of RNA hydrolysis are used (not RNA per se as a certain sequence or secondary configuration). A highly purified yeast-derived RNA with homogeneous molecular weight was suggested for the treatment of inflammation and inflammation-related disorders. (Tkachuk Z., Compound, composition and method for the treatment of inflammatory and inflammatory-related disorders, U.S. Pat. No. 6,737,271, May 18, 2004) Based on this substance, the therapeutic agent Nucleinat was created. It has successfully completed phase 2 of clinical evaluation and has proved to be an effective anti-inflammatory agent for the treatment of acute and chronic pulmonary inflammatory disorders, inflammations of kidneys, and other inflammatory diseases. However, highly purified yeast-derived RNA is devoid of specific antiviral activity. Till now nucleic acids, and, in particular, RNAs have not been used as specific antiviral agents with multiple antiviral action.
The Influenza Viruses.
Influenza virus belongs to the orthomyxoviridae family, and has three serotypes: A, B and C. Serotype A and B viruses belong to the same genus, while serotype C viruses represent a different genus. Each serotype is characterized by its own set of antigenic characteristics which are determined by nucleoproteins (NP) and matrix (M) protein antigens. Type A influenza viruses are widely prevalent in nature and can affect humans as well as some mammals and birds. Type B influenza viruses are isolated only from humans, while type C viruses can be isolated from both humans and pigs. Type A and B viruses are responsible for the yearly flu epidemics. Serotype A consists of subtypes which are characterized by different hemagglutinin (H) and neuraminidase (N) properties. Serotype A viruses and, to a lesser extent, serotype B viruses are characterized by frequent variations of antigenic structure under natural conditions. Antibody response to subtype-specific hemagglutinin is the cornerstone of influenza immunity. Currently there are 15 known subtypes of hemagglutinin (H) and 10 subtypes of neuraminidase (N) of type A influenza viruses circulating in vertebrates. Virological, immunological and seroarcheological studies show that since 1889 epidemics and pandemics have been caused by viruses containing hemagglutinin H1, H2 or H3, and neuraminidase N1 or N2. These viruses have been classified into three subtypes of human A influenza virus designated as A (H1N1), A (H2N2), and A (H3N2). They are responsible for influenza outbreaks with predictable cyclic recurrences of the same virus strains. Viruses replicate in the epithelium of nasopharyngeal and upper respiratory tract mucosa, and produce a potent toxin affecting blood vessels and capillaries.
During epidemics, the virus of influenza attacks a great number of people, influencing negatively the economies of countries. Therefore, search for and the use of chemotherapeutic drugs capable of blocking the virus reproduction becomes very important. Until recently, effective chemoprophylaxis of influenza, as well as other virus diseases was very complicated. The first mentioning on antiviral action of 1-amino-adamantane was done in 1963 by Jackson and co-authors. (Jackson G. G., Muldon R. L., Akers L. W., Serological evidence for prevention of influenza infection in volunteers by anti-influenzal drug amantadine hydrochloride, in Antimicrobial agents and chemotherapy, S. I., Acad. Press, New York, 1964, pp. 703-707). In 1964 Device and co-authors published the results of experiments (Davies W. L., Grunert R. R., De Somer P. et al., Antiviral activity of 1-adamantanamine (amantadine), Science, 1964, 144, pp. 862-863) Remantadine became the first drug, which was widely used for the prevention and treatment of influenza.
In 1970, the crystal structure of viral neuraminidase of flu A and B types was discovered and it was proved that inhibiting the neuraminidase component of influenza virus delays the reproduction of virus. (Miller W. E., Mechanisms of action and pharmacology chemical agents, in Antiviral agents and viral diseases of man, Ed. by G. J. Gallasso et al., Raven Press, New York, 1979, pp. 77-149; Hay den F. G., Osterhaus A. D., Treanor J. J. et al., Efficacy and safety of the neur-aminidase inhibitor zanamivir in the treatment of influenza virus infections, in GG 167 Influenza Study Group, N. Engl. J. Med., 1997, 337, 13, pp. 874-880) This allowed creating drugs that block the activity of neuraminidase of A and B virus types—oseltamivir and zanamivir. These drugs protect from infecting the epithelial cells of respiratory tract and prevent spreading of the virus in body. Oseltamivir and zanamivir showed high preventive and treatment effectiveness with a decreased period of treatment by 2-3 days on average and an easier course of disease. [Monto A. S., Fleming D. M., Henry D. et al., Efficacy and safety of the neuraminidase inhibitor zanamivir in the treatment of influenza A and B virus infections, J. Infect. Diseases, 1999, 180, 2, pp. 254-261; Iozzo M., Efficacy and tolerability of the neuraminidase inhibitor Zanamivir, J. Acad. Phys. Assistants, 2001, 2, 2, pp. 186-188; Hoyden F. G., Treanor J. J., Fritz R. S. et al., Use of oral neuraminidase inhibitor Osettamivir in experimental human influenza, J. Amer. Med. Assoc., 1999, 282, 13, pp. 48-50)
The epidemics of influenza and acute respiratory virus infections are accompanied by high death rate and serious complications in the vulnerable population groups. The risk group includes people with chronic conditions, especially cardio-vascular diseases. (Davis M. M., Taubert K., Benin A. L. et al., Influenza vaccination as secondary prevention for cardiovascular disease: a science advisory from the American Heart Association, Amer. College of Cardiology, Circulation, 2006, 114, pp. 1549-1553)
There is convincing evidence that acute respiratory virus infections and influenza cause heart attacks and it was proved that drugs against viral infections are an effective method for decreasing the risk of heart attacks in people with cardio-vascular diseases. (Warren-Gash C, Smeeth L., Hayviard A. C., Influenza as a trigger for acute myocardial infarction or death from cardiovascular disease: a systematic review, Lancet Infect. Dis., 2009, 9, pp. 601-610)
Epidemics of influenza and ARVD are accompanied by heavy mortality and dangerous complications among vulnerable set of men. To the population at risk belong people with chronic pathologies, especially with such as cardiovascular diseases. (Davis M. M., Taubert K., Benin A. L. et al., Influenza vaccination as secondary prevention for cardiovascular disease: a science advisory from the American Heart Association, American College of Cardiology, Circulation, 2006, 114, pp. 1549-1553) Yet, the connection between contagion with ARVD and influenza and the acute myocardial infarction remained unclear for long. Different works reported the seasonality of cardiovascular mortality models, what reminded models of ARVD and influenza circulation. (Ailing D. W., Blackwelder W. C., Stuart-Harris C. H., A study of excess mortality during influenza epidemics in the United States, 1968-1976, Am. Epidemiol., 1981, 113, pp. 30-33; Collins S. D., Excess mortality from causes other than influenza and pneumonia during influenza epidemics, Public Health Rep., 1932, 47, pp. 2159-2179; Eickhoff T., Sherman I, Serfling R., Observations on excess mortality associated with epidemic influenza, JAMA, 1961, 176, pp. 776-782; Housworth J., Langmuir A. D., Excess mortality from epidemic influenza, 1957-1966, Am. J. Epidemiol., 1974, 100, pp. 40-48)
Clinical scores in patients with influenza pointed to the definitive systemic impacts, such as high temperature, muscle pain and fatigue, and also indicated frequent myocardial ischemia episodes. (Greaves K., Oxford J. S., Price C. P., Clarke G. H., Crake T., The prevalence of myocarditis and skeletal muscle injury during acute viral infection in adults: measurement of cardiac troponins I and T in 152 patients with acute influenza infection, Arch. Intern. Med. 2003, 163, pp. 165-168; Ison M. G., Campbell V., Rembold C., Dent J., Hayden F. G., Cardiac findings during uncomplicated acute influenza in ambulatory adults, Clin. Infect. Dis., 2005, 40, pp. 415-422; Paul B. K., Clinical observations of influenza, with special reference to its effects and cardiac functional efficiency, Indian Med. J., 1963, 57, pp. 251-283; Verel D., Warrack A. J., Potter C. W., Ward C., Rickards D. F., Observations on the A2 England influenza epidemic: a clinicopathological study, Am. Heart J., 1976, 92, pp. 290-96) These results led to the inference that influenza can play a role of an acute inflammatory stimulus, which triggers cardiovascular incrusions. In order to prognose risk of vascular diseases in humans markers of systemic inflammation and inflammatory cells activation came to be used as an important component of
It is known that influenza virus greatly affects inflammatory, coagulation and metabolic pathways (Madjid M., Aboshady I., Awan I., Iitovsky S., Casscells S. W., Influenza and cardiovascular disease: is there a causal relationship, Tex. Heart Inst. J., 2004, 31, pp. 4-13), which can lead to atheromatous plaque destabilization and thereby to the partial occlusion or the complete obturation of the coronary vessel, which is the main cause of the acute infarction. (White H. D., Chew D. P., Acute myocardial infarction, Lancet, 2008, 372, pp. 570-584) Moreover, influenza can act as an acute inflammatory and pro-coagulator stimulus of the rapid changes in the endothelium. (Housworth J., Langmuir A. D., Excess mortality from epidemic influenza, 1957-1966, Am. J. Epidemiol. 1974, 100, pp. 40-48; Madjid M., Awan I., Ali M, Frazier L., Casscells W., Influenza and atherosclerosis: vaccination for cardiovascular disease prevention, Expert Opin. Bid. Ther., 2005, 5, pp. 91-96) Well known are heart complications as a result of influenza infection, such as myocarditis, pericarditis, yet the influenza role under the character of the trigger mechanism of acute myocardial infarction is not clearly proved. At the same time in the task-specific literature emerged many facts of that the influenza (including influenza as a disease and a acute respiratory infection) can precipitate acute myocardial infarction or even vascular death. Numerous investigation, conducted at different conditions, using different methods indicate that there is a tight connection between influenza and acute myocardial infarction. At the same time there are not many clinical studies, which would investigate the possibility of risk reduction of cardiovascular complications by using antiviral and cardioprotective medications in patients with influenza and ARVD. Only in two small randomized investigations was estimated a positive impact of anti-influenza vaccination on the prevention of heart seizures in patients with cardiovascular diseases and it was shown that vaccination against influenza results in considerable protection against vascular death. Obtained summarized valuation of the model of randomized consequences intends whilst insignificant, still protective effect of vaccination. Authors consider that in cases, where it was shown, anti-influenza vaccination should be encouraged, especially in people with cardiovascular diseases, which are often of prejudged oppositional code of vaccination. (Warren-Gash C, Smeeth L, Hayviard A C. Influenza as a trigger for acute myocardial infarction or death from cardiovascular disease: a systematic review, Lancet Infect. Dis., 2009, 9, pp. 601-610)
Thus, there is a luculent proof of the fact that influenza can provoke development of acute myocardial infarction, increase vascular death, but the usage of anti-Viral medications can be an effective way to reduce the rick of heart seizures in patients with cardiovascular diseases.
The Hepatitis Viruses
According to World Health Organization (WHO), hepatitis is defined as inflammation of the liver caused by infectious or toxic agents. In most cases it is caused by one of the five viral agents: hepatitis A virus (HAV), hepatitis B virus (HBV), hepatitis C virus (HCV), the HBV-associated delta agent or hepatitis D virus (HDV), and hepatitis E virus (HEV).
Hepatitis A is an acute infectious disease predominantly affecting the gastrointestinal tract, particularly the liver. Hepatitis A is caused by hepatitis A virus which is spread through contaminated food or water. Persons with acute hepatitis A are the source of infection. The disease is dangerous as it leads to liver cell death and liver function impairment.
Hepatitis B is caused by hepatitis B virus (HBV) which affects the liver by causing its inflammation. About 600,000 people worldwide die each year of hepatitis B-related complications. HBV is transmitted from infected persons through blood or other body fluids like semen, vaginal secretions, and saliva. The modes of transmission of hepatitis B virus are the same as those of human immunodeficiency virus (HIV), but compared with the latter the HBV infectivity is 50-100-fold higher. Unlike HIV, HBV can resist in the environment beyond human organism for at least 7 days.
Hepatitis D. Hepatitis D is caused by hepatitis D virus (or delta virus) and is characterized by acute onset with massive liver cell damage. Delta virus is unable to replicate on its own, it requires the presence of HBV for its replication and expression. Hepatitis D follows a similar mode of transmission as hepatitis B and hepatitis C. Its incubation period is 3 to 7 weeks. Clinical manifestations of hepatitis D are similar to those of hepatitis B. Vaccination against hepatitis B also prevents hepatitis D.
Hepatitis E. Hepatitis E is similar to hepatitis A in its symptoms. The modes of transmission of hepatitis E are also similar to those of hepatitis A. Hepatitis E is spread through contaminated water or food, and can also be transmitted through blood. It occurs primarily in Central Asia and in African countries.
Other agents, such as hepatitis F virus and hepatitis G virus have been recently identified, but as yet have not been studied extensively.
Hepatitis C is a viral infection with parenteral mode of transmission occurring most frequently in the form of post-transfusion hepatitis. HCV was discovered in 1989 in patients with hepatitis whose blood contained a viral RNA similar in organization to that of flaviviruses. Hepatitis C is caused by an RNA-containing virus of the family Flaviviridae. Its virion is estimated to be 30 to 60 nm in diameter. To date up to 11 distinct genotypes of HCV have been identified: 1a, 1b, 1c 2a, 2b, 2c 3a, 3b 4a, 4b, 4c, 4d, 4e 5a 6a 7a, 7b 8a, 8b 9a 10a 11a. HCV consists of structural (capsid and envelope) and non-structural viral proteins, and tends to circulate in blood in low titer. It is associated with low density lipoproteins and antibodies to HCV proteins. The sources of infection are patients with manifest and latent forms of hepatitis C. The virus is transmitted by parenteral route through blood and blood products. The current standard of treatment of hepatitis C, approved in a number of countries, is a combination antiviral therapy with alpha interferon and ribavirin. It is indicated in HCV-RNA-positive patients with persistently elevated serum ALT levels and marked histological liver damage. The duration of therapy may range from 12 to 72 weeks, depending on HCV genotype and on the response to treatment determined by the individual patient characteristics and host's genome. The current criteria of effectiveness of hepatitis C therapy include a sustained biochemical remission (persistent normalization of alanine aminotransferase levels after antiviral therapy), and absence of viremia (i.e., clearance of HCV RNA at 6 months after the end of therapy).
Hepatitis C Virus is a global health problem. Its agent is an RNA-containing virus, which is transmitted orally and causes an acute or permanent hepatitis, which can lead to cirrhosis or liver cancer in 60-80% cases. In accordance with the information of WHO, about 1% of world population are infected with Hepatitis C Virus (HCV). The source of infection is a human; in 100% of cases, the virus can be found in the blood of an infected patient. Hepatitis C Virus is called <<the silent killer>>. In 70% of cases the illness is taking a latent form. Regardless of the severance, in 50-80% cases Hepatitis C Virus transforms into a permanent illness with the consequential development of cirrhosis, carcinoma, damages neural cells, causes severe consequences.
The virus is categorized under the type Hepatitis C-like viruses of the Flaviviridae family (Alter Y. J., To or not to C: these are questions, Blood, 1995, 85, pp. 1681-1695; National Institutes of Health Consensus Development Conference Panel Statement: Management of Hepatitis C, Hepatology, 1997, 26, pp. 2-10), contains a positive polarity RNA (Lindsay K. L., Therapy of hepatitis C overview, Hepatology, 1997, 26, pp. 715-775), which has a high degree of heterogeneity. (Yohko K. S., Hiroshi Y., In vitro systems for the detection of hepatitis C virus infection, Viral Hepatitis Rev., 1995, 1, pp. 59-65; National Institutes of Health Consensus Development Conference Panel Statement: Management of Hepatitis C, Hepatology, 1997, 26, pp. 2-10) α-Interferon remedies (IFN)—reaferon, roefron A, intron A, and others—remain to be the basis for the treatment of HCV-infection (EASL International Consensus Conference on Hepatitis C. Consensus Statement. Paris, J. Hepatol., 1999, 30, pp. 976-995), however, the reliable anti-virus effect (absence of HCV RNA in the blood serum 6 month after the treatment) can be observed only in 8-12% of patients treated with a-Interferon. (Lindsay K. L., Therapy of hepatitis C^overview, Hepatology, 1997, 26, pp. 715-775) Therefore, the search for alternative ways of therapy of the HCV infection remains important nowadays.
IFN inductors, which represent a diverse group high- and low-molecular natural and synthetic compounds, which are related by their ability to cause IFN production, is a perspective group for the treatment of virus infections, including HCV. IFN inductors have typical, for IFN, antivirus, antiproliferative, and immune-modulating activities (Ershov F. Y., Anti-virus Pharmaceuticals, Moscow, 1998, p. 240)
The vaccine against HCV has not been developed yet, and there are no effective drugs capable of inhibiting the virus replication in body. The difficulties with finding preventive and treatment medications against HCV are related to the unsuccessful attempts to obtain an experimental model of the virus required for screening tests for identification of the drugs, which can effectively treat HCV.
Herpes viruses
Herpesvirus (Latin Herpesviridae) is a large family of DNA-containing viruses that cause various diseases not only in humans and other mammals, but birds, reptiles, amphibians, fish. Herpes viruses infected the majority of the population of our planet. (Baltimore D. The strategy of RNA viruses, Harvey Lect., 1974, 70 Series, pp. 57-74)
Herpetic infection is a classic example of a latent infective process. Above 70% of patients suffered recurrences of infection after the first contact with herpes simplex virus (HSV) regardless of high antiherpetic antibody levels. Herpetic infections represent a group of anthropozoonotic infectious diseases caused by human herpes viruses (HHV) with varying types of clinical course, comprising unapparent, subclinical and manifest clinical forms. (Liesegang T., Herpes simplex, Cornea, 1999, Vol. 18, No. 6., p. 739)
Herpes (Greek origin meaning “creeping”) is one of the most prevalent and poorly controlled human infections. In a host with the normal immune system herpetic viruses may circulate asymptomatically, while in immunocompromised individuals they may cause severe potentially lethal. In humans 8 types of herpes viruses have been identified: these are represented by DNA-containing viruses with similar morphology which are indistinguishable on electron microscopy. (Herpesviruses, in: Baron's Medical Microbiology, Baron S et al., Eds., 4th.; U. of Texas Medical Branch, 1996; Medical Microbiology, 5th Ed., Elsevier Mosby, 2005) Entering a human organism, a herpes simplex virus (HSV) infects its host forever causing occasionally recurrences of varying. HSV in its latent state is localized in paravertebral sensor ganglia in the form of L-PREP-particles.
The most common human herpes virus (HHV) is the type 1 herpes simplex virus, or HSV-1, causing orofacial herpes. Similar morphological, antigenic, chemical, and physical properties are shared by type 2 human herpes virus (HSV-2 or HHV-2) causing genital herpes. Genital herpes (GH) is a particular herpetic infection, representing one of the most prevalent sexually transmitted disease. Its prominent feature is that the causative agent will remain with the carrier for the rest of his/her life (latency). This is the reason for the high incidence and frequent recurrences of the disease.
The onset of GH is often associated with HSV-2. This is supported by the high incidence of antibodies (Ab) to this virus serotype found in epidemiological. Previously it has been considered that HSV-1 is more frequently identified in skin infections involving face, trunk and upper limbs. Now it is well established that GH may be caused by HSV-1. HSV-1-caused GH is characterized by low recurrence rates, the recurrences are more frequently observed in patients with high titers of anti-HSV-2 antibodies. This virus plays a role in pathogenesis cervical cancer as demonstrated by means of hybridization of HSV DNA with the DNA of tissues obtained during surgeries for cervical cancer and cervical canal malignancies.
Type 3 human herpes virus (HHV-3) causes two different clinical entities—varicella zoster and herpes zoster. Type 4 human herpes virus (HHV-4), or Epstein-Barr virus, is a causative agent of infectious mononucleosis, Burkitt's lymphoma, nasopharyngeal carcinoma, hairy tongue leukoplakia. Type 5 human herpes virus (HHV-5) causes cytomegalovirus infection, and finally, type 6 human herpes virus (HHV-6), according to recent studies, is associated with exanthema subitum in infants and chronic fatigue syndrome in adults. Current literature reports indicate that HHV-6 may play a role in development of lymphogranulomatosis, malignant. B-cell lymphoma, sarcoidosis, Sjogren's syndrome, Crohn's disease, autoimmune thyroiditis, non-Epstein-Barr virus infectious mononucleosis. It may cause acute hepatitis in children and adults, including fulminating hepatitis with lethal outcome. In 1990 HHV-7 and HHV-8 were discovered, their role is yet to be established. HHV-7 is associated with lymphoproliferative disorders and chronic fatigue syndrome, while HHV-8 is associated with Kaposi sarcoma.
Treatment of herpetic infection is still challenging. A long-lasting chronic process leads to negative regulation of the immune system. Secondary immunodeficiency, suppression of cell-immunity and decrease of non-specific resistance are observed, manifesting as decreased ability of white blood cells to synthesize α- and γ-interferons (IFN), hypoimmunoglobulinemia, sensitization to viral antigens. (Herpesviruses, in Baron's Medical Microbiology, Baron S et al., Eds.; 4th U. of Texas Medical Branch, 1996; Medical Microbiology, 5th Ed., Elsevier Mosby, 2005)
Currently antiherpetic drugs constitute around 80% of the available antiviral agents re-emphasizing the importance of the problem. Most of them are represented by anomalous nucleosides. The mechanism of their action combines the inhibition of enzymes involved in viral replication (thymidine kinase, DNA-polymerase) with the induction of IFN synthesis. Hence is the question: why despite a significant number and variety of antiviral agents, herpetic infections are still poorly controlled? Recent reports indicate that resistance of HSV to antiherpetic anomalous nucleosides is increasing over the past years.
However the most efficient way is the development of antiviral agents capable of affecting the early stages of viral replication, namely adsorption and merging with cells. Novel antiviral agents are being modeled as ligand imitators or receptor imitators capable of competitive substitution of the natural components while interacting with the host cell. The aim of the present study was to assess the efficacy of antiviral action of RNA preparations possessing the above-mentioned properties.
Human Immunodeficiency Virus
The first case of acquired immunodeficiency syndrome (AIDS) was reported in the USA in 1983. The patient died 2 months later. Currently around 14,000 of new infections occur daily. The causative agent is a virus with a helical structure within a triangular core. It is known as human immunodeficiency virus (HIV) and has three types: HIV-1 and HIV-2 which are highly prevalent in Western Europe, and HIV-3 which is prevalent mostly among Africans and Americans. The virus infects T-lymphocytes which serve for its replication, and macrophages which spread the virus through the organism. AIDS per se is not a fatal disease, but HIV down regulates the immune system of the human organism so that even a common cold may lead to death. HIV destroys T-lymphocytes and the human organism loses its defense mechanisms resulting in increased vulnerability to ordinary infections. The risk of fatal infections, nervous system involvement, and cancer increase dramatically. The source of infection is an HIV carrier. Transmission is possible during sexual intercourse with an HIV-infected person or by blood to blood contact. If an infected mother gives birth to a child, the child is not necessarily a virus carrier. Antiretroviral therapy permits to decrease the risk of mother-to-child transmission to as low as 6 percent. (Sepkowitz K. A., AIDS—the first 20 years, N. Engl. J. Med., 2001, 344, 23, pp. 1764-72; Divisions of HIV/AIDS Prevention HIV and Its Transmission. Centers for Disease Control &Prevention, 2003) The first antiretroviral drug known as azidothymidine was first synthesized in 1964, and in 1987 it was approved for the treatment of HIV-infection and has been widely used as an antiretroviral agent since then. (Balzarini J., Naesens L., Aquaro S., Knispel T., Pemo C.-F., De Clercq E., Meier C., Intracellular metabolism of cyclosaligenyl 3′-azido-2′-3′-dideoxythymidine monophosphate, a prodrug of 3′-azido-2′-3′-dideoxythymidine
(Zidovudine), Molecular Pharmacology, 1999, 56, pp. 1354-1361) In the mid-90s, the first protease inhibitors became available, including saquinavir, ritonavir, and indinavir. Their use permitted to decrease mortality from 38 to 22 percent. (Cameron D. W., Heath-Chiozzi M., Danner S., Cohen C, Kravcik S., Maurath C., Sun E., Henry D., Rode R., Potthoff A., Leonard J., Randomised placebo-controlled trial of ritonavir in advanced HIV-1 disease, The Advanced HIV Disease Ritonavir Study Group, Lancet, 1998, 351, 9102, pp. 543-549) In 1996 the first non-nucleoside reverse transcriptase inhibitor nevirapine and another protease inhibitor nelfinavir were introduced. In Europe, the use of new drugs permitted to decrease the AIDS-related morbidity from 30.7 to 2.5 percent. (Harrington M., Carpenter C. C., Hit HIV-1 hard, but only when necessary, Lancet, 2000, 355, 9221, pp. 2147-52)
However, despite a high number of quiet expensive antiretroviral agents, there is a growing problem of HIV multidrug resistance. This re-emphasizes the importance of search for new agents targeting at multiple HIV proteins. Of particular importance is the need of new antiviral drugs capable of inhibiting replication of not only HIV, but also of other viruses, e.g., hepatitis viruses which frequently represent a concomitant infection in HIV-positive patients.
Enteroviruses belong to the Picornaviridae family. They encompass 67 serotypes which are pathogenic for humans: 3 serotypes of poliovirus, 23 serotypes of coxsackievinis A, 6 serotypes of coxsackievirus B, and others. Enteroviruses are so named because of their ability to multiply in the gastrointestinal tract. Despite their name, these viruses rarely cause prominent enteritis. Human enteroviruses contain a single-stranded RNA encoding a polyprotein that is cleaved into 11 different proteins. The enteroviral RNA genome is surrounded by an icosahedral capsid comprising four viral proteins (VP1-VP4). VP1 is the predominant target neutralizing antibodies. Enteroviruses have an ubiquitary distribution. Coxsackieviruses are responsible for a broad spectrum of diseases in humans, most commonly, aseptic meningitis and myalgia. These viruses are spread by the fecal-oral route as well as the other enteroviruses. Coxsackieviruses are divided into group A and group B. Group A includes about 24 serotypes causing herpangina, aseptic meningitis, pericarditis, nonspecific febrile illness, and others. Group B is responsible for Bornholm disease (pleurodynia, myalgia). Diseases associated with group A and group B coxsackieviruses have distinct clinical features.
Coxsackievirus B4 affects both central and peripheral nervous system, causing headache (particularly, occipital headache) and chest pain.
The need for new antiviral agents.
Currently, there is a need of development of novel antiviral agents with multiple action against various viral diseases. These agents should interfere with the mechanisms responsible for viral penetration into the host cell and viral release.
The recognition of the possibility of an antibiotic drug, capable of inhibiting many representatives of the virus family was based on the discoveries by Charles Janeway and his followers. (Janeway Jr., C. A., Approaching the asymptote? Evolution and revolution in immunology, Cold Spring Harbor Symposia on Quantitative Biology, 1989, 54, 1, pp. 1-13) First, it was shown that all live organisms possess an innat immunity. This innat immunity exists on the genetic level and provides for the ability of organism to fight alien microorganisms, transplants, toxins, tumor cells, and cells infected with viruses. The system of innat immunity is activated at first emergence of a pathogen and reacts at certain classes of antigens specific for pathogen organisms. Second, the cells of innat immunity possess the receptors called Toll-like receptor, TLR, which recognize these classes of antigens and activate the innat immunity. Third, the TLR is specific to certain class of infections, i.e., is capable to recognize specific type of alien structures, for example, virus infection RNA, rather than general. People have more than ten TLRs, each specializing in certain class of pathogens. One recognizes RNA of virus infections, another—polysaccharides of bacteria, another—proteins of single-cell parasites, etc. The
Receptors are located on different types of cells, including the cells of skin and epithelium.
In case of recognition of virus factor, the infected cell with the help of TLR can switch off the synthesis of virus protein, to initiate the programmed death of infected cell (apoptosis). Immune cells which identified the virus can spread signals for the expression of cytokines, factors causing inflammations, and also can emit antivirus factors such as interferon.